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Subunits of the isolated K+ and Cl channel protein from ehrlich ascites tumour cells
Abstracts A9 =$EC’$S OF EXTRACELLULAR SODIUM ANDpH ON Na /‘Ii EXCHANGE IN CRAYFISHNEURONS. S. Gaillard and J.-L. Rodeau, Lab. d’Etude des R&ulations Physiologiques, CNRS, Strasbourg, France. The amiloride-sensitive component of intracellular pH (PH.) regulgtion in crayfish neyrons was studied. at 20 C with pH- and Na selective microelectrodes, as a function of extracelluiar pH (pH ) and sodium concentration ([Na ] ) in bycarbonate-free media. At PH = 7.5 anb [Na+] - 200 mM, the steady s&e PH. was 7.09kg.12 and the rate constant of the eiponential PH. recgyery after an acid load was 0.081+0.02& min . The rate of recovery depended on [Na+] (range 5-200 mM) according to MichaeliseMenten kinetics with an apparent Km of 39+10 mM, and on pH (range 6.5-8.5) according to a simple titratfon curve with an apparent pK of 7.05kO.21. Decreasing [Na+]_ below 30 mMor pH_ below 6.9 resulted in a”decrease of the &8agy Ttate pHi. The kinetic properties of the Na /H exchange in crayfish neurons are similar to those described in vertebrate cells. Variations of intracelluar Nal with pH terms of pH sensitivity gf
809
A 10 ~&l’ROPHYSIOLOG~C EVIDENCE FOR INDEPENDENT ACTIVATION OF K ANDCl CONDUCTANCES DURING THE REGULATORY VDLUMEDECREASEIN CULTURED EPITHELIAL CELLS. A. Hazama and Y. Okada, Dept. of Physiology, Faculty of Medicine, Kyoto Univ. Kyoto 606, Japan.
During exposure to a hypotonic solution, an intestinal epithelial cell line (Intestine 407) exhibited a regulatory volume decrease (RVD) after initial osmotic swelling. To obtain direct evidence for the involvement of electroconductive ion transport pathways in the RVD process, a variety of electrophysiological approaches, such as conventional intracellular recordings, two-microelectrode voltage clamp studies, tight-seal whole-cell recordings and ion-selective microelectrode studies, were made in the present study. Based on these data, it is concluded that qparable two conductive pathways, that is, K and Cl- channels, are operating in the volume regulation.
A 12 A 11 SODIUM-COUPLEDGLYCINE UPTAKE BY EHRLICH SUBUNITSOF THE ISOLATEDK+ AND Cl- CHANNEL CELLS. CELLSRESULTSIN AN INCREASEIN CELL VOLUMEPROTEINFROMEHRLICHASCITES TUMOUR F. Jessen and E.K. HOffIIBM, August Krogh ANDMEMBRANE CONDUCTANCE TO Cl AND K. Institute, Univ. of Copenhagen, DK-2100 0. R.L. Hudson and S.G. Schultz, Dept. of Physiology and Cell Biology, Univ. of Texas. Conductive K+ and Cl- channels are activated Medical School, Houston, TX 77225, USA. during regulatory volume decrease in Fhrlich cells. We have purified membrane proteins by Sodium-coupled glycine uptake by Ehrlich aschromatography using furosemide (an cites tumor cells results in an increase in affinity inhibitor of the chloride channel) as ligand. Studies employing the patchcell volume. clamp technique in the cell-attached mode Patch-clamp measurements on the isolated prointo lipid bilayers shows indicate that within 0.5-3 minutes after the tein reconstituted the existence of both a conductive K+ and a activation of this Na-coupled uptake process conductive Cl- channel (B.D. Cherksey et al. there is a marked increase in single-channel 1987, J. Physiol., Land. (in the Press)). activity. Studies on excised patches indicate that one of these channels conducts Cl. The Analysing the isolated proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results of whole-cell current clamping indiunder reducing conditions we find two domicate that the activation of this cotransport nating bands of m.w. 95 kDa and 43 kDa. Perprocess results in an initial, rapid depolaforming native PAGEin the presence of chorization of the electrical potential diffewe find three major bands of about 250 rence across the membrane followed by a spon- late kDa, 200 kDa and 60 kDa. SDS-PAGE of the partial repolarization. The initial taneous, depolarization can 9 attributed to the rheo- three individual proteins under reducing congenie entry of Na coupled to the entry of ditions shows that the 250 kDa and the 200 glycine; the spontaneous repolarization is kDa bands mainly consist of a 95 kDa subunit almost certainly the result of an increase in and that the 60 kDa protein in SDS runs as a 43 kDa protein. Conclusion: the 95 kDa and the conductance of the plasma membrane to K. the 43 kDa polypeptides seems to originate from two separate proteins.
809
A 10 ~&l’ROPHYSIOLOG~C EVIDENCE FOR INDEPENDENT ACTIVATION OF K ANDCl CONDUCTANCES DURING THE REGULATORY VDLUMEDECREASEIN CULTURED EPITHELIAL CELLS. A. Hazama and Y. Okada, Dept. of Physiology, Faculty of Medicine, Kyoto Univ. Kyoto 606, Japan.
During exposure to a hypotonic solution, an intestinal epithelial cell line (Intestine 407) exhibited a regulatory volume decrease (RVD) after initial osmotic swelling. To obtain direct evidence for the involvement of electroconductive ion transport pathways in the RVD process, a variety of electrophysiological approaches, such as conventional intracellular recordings, two-microelectrode voltage clamp studies, tight-seal whole-cell recordings and ion-selective microelectrode studies, were made in the present study. Based on these data, it is concluded that qparable two conductive pathways, that is, K and Cl- channels, are operating in the volume regulation.
A 12 A 11 SODIUM-COUPLEDGLYCINE UPTAKE BY EHRLICH SUBUNITSOF THE ISOLATEDK+ AND Cl- CHANNEL CELLS. CELLSRESULTSIN AN INCREASEIN CELL VOLUMEPROTEINFROMEHRLICHASCITES TUMOUR F. Jessen and E.K. HOffIIBM, August Krogh ANDMEMBRANE CONDUCTANCE TO Cl AND K. Institute, Univ. of Copenhagen, DK-2100 0. R.L. Hudson and S.G. Schultz, Dept. of Physiology and Cell Biology, Univ. of Texas. Conductive K+ and Cl- channels are activated Medical School, Houston, TX 77225, USA. during regulatory volume decrease in Fhrlich cells. We have purified membrane proteins by Sodium-coupled glycine uptake by Ehrlich aschromatography using furosemide (an cites tumor cells results in an increase in affinity inhibitor of the chloride channel) as ligand. Studies employing the patchcell volume. clamp technique in the cell-attached mode Patch-clamp measurements on the isolated prointo lipid bilayers shows indicate that within 0.5-3 minutes after the tein reconstituted the existence of both a conductive K+ and a activation of this Na-coupled uptake process conductive Cl- channel (B.D. Cherksey et al. there is a marked increase in single-channel 1987, J. Physiol., Land. (in the Press)). activity. Studies on excised patches indicate that one of these channels conducts Cl. The Analysing the isolated proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results of whole-cell current clamping indiunder reducing conditions we find two domicate that the activation of this cotransport nating bands of m.w. 95 kDa and 43 kDa. Perprocess results in an initial, rapid depolaforming native PAGEin the presence of chorization of the electrical potential diffewe find three major bands of about 250 rence across the membrane followed by a spon- late kDa, 200 kDa and 60 kDa. SDS-PAGE of the partial repolarization. The initial taneous, depolarization can 9 attributed to the rheo- three individual proteins under reducing congenie entry of Na coupled to the entry of ditions shows that the 250 kDa and the 200 glycine; the spontaneous repolarization is kDa bands mainly consist of a 95 kDa subunit almost certainly the result of an increase in and that the 60 kDa protein in SDS runs as a 43 kDa protein. Conclusion: the 95 kDa and the conductance of the plasma membrane to K. the 43 kDa polypeptides seems to originate from two separate proteins.